This subproject is one of many research subprojects utilizing the resources provided by a Center grant funded by NIH/NCRR. The subproject and investigator (PI) may have received primary funding from another NIH source, and thus could be represented in other CRISP entries. The institution listed is for the Center, which is not necessarily the institution for the investigator. Mimivirus is a recently identified parasite of Acanthamoeba polyphaga that has an extraordinarily large genome (1.2 Mb) encoding 911 predicted proteins. The mimivirus proteome embraces a slew of enzymes with imputed functions in DNA and RNA metabolism. Mimivirus appears to encode its own mRNA synthetic machinery, which includes a multisubunit RNA polymerase and a putative RNA capping enzyme that resembles the vaccinia virus D1 protein. These features highlight the likelihood that the mimivirus gene expression strategy will have a poxvirus-like flavor. We therefore examined the biochemical properties of the mimivirus capping enzyme and the extent to which it resembles or differs from the vaccinia capping machinery. We have purified, assayed, and crystallized residues (1-237) of the tri-functional mimivirus mRNA capping enzyme which comprises a minimal autonomous triphosphatase domain. Alanine substitutions at each of the four predicted metal-binding glutamates (Glu37, Glu39, Glu212, Gl214) abolished the NTP phosphohydrolase activity of MimiCE, thereby verifying its membership in the metal-dependent viral/fungal/protozoal triphosphatase family. Thus, the small triphosphatase domain of the mimivirus capping enzyme (which we can purify from bacteria in 50 mg quantities) presents a tractable target for the investigation of viral metal-dependent RNA triphosphatase proteins. Based on extensive similarities between poxvirus capping enzymes and that of mimivirus, the observations gained in the study of the mimivirus capping triphosphatase should be applicable to poxvirusus at large and, therefore, of great use in structure-facilitated drug design.